Genetic inheritance, and resistance to age-related diseases such as cancer, depend on the stability of the human genome. Stable genome maintenance in turn depends critically on cellular systems that process "spontaneous" DNA damage. Such damage includes hydrolytic decay lesions, in particular abasic sites, as well as lesions formed by metabolic by-products such as free radicals and alkylating agents. Existing data indicate that the base excision repair (BER) system is most likely to deal with this endogenous DNA damage. In BER, DNA glycosylases remove altered bases to generate abasic sites, which are further processed in several steps dependent on the Ape1 endonuclease and DNA polymerase beta. Our long-term goal has been to ascertain the biological functions of Ape1 protein as a central player in base excision repair (BER) of DNA and perhaps other processes, and to understand the coordination of activities in the various branches of BER. Past efforts have been valuable in defining the biochemistry of Ape1 activity, but only recently have we been able to conduct genetic tests of the protein's cellular function through the use of small-interfering RNA (siRNA). These data clearly support an essential role of Ape1 in maintaining the viability of human cells by processing abasic DNA damage formed by endogenous processes. Other recent work points to a novel role for the sirtuin Sirt6 in modulating BER, possibly through effects on the activity of DNA polymerase beta. Since deletion of the SIRT6 in mice shortens lifespan and generates various phenotypes associated with premature aging, these observations provide the first direct link between BER and the normal aging process. The work proposed here builds on this success in order to define the cellular function of Ape1 and the origins of the DNA damage that it processes, and how the BER can be modulated by a sirtuin. Three specific aims will address our goals: 1. Establish human or mouse cell lines with regulated expression of APE1-specific si-RNA and examine the kinetics of the biological effects accompanying Ape1 depletion; 2. Determine the contribution of DNA glycosylases (in particular UNG2, OGG1, AAG/MPG, and NTH1) and of oxidative damage as sources of the lethal lesions that accumulate in Ape1-deficient cells; 3. Directly test the role of SirtG in DNA repair and the sub-pathways of BER.